Quantifying and Identifying the Active and Damaged Subsets of Indigenous Microbial Communities

Abstract

Flow cytometry and fluorescent dyes represent valuable experimental tools for studying complex microbial communities, enabling the quantification and sorting of cells with distinct levels of activity or damage, and providing information that can be difficult to infer from metagenomic sequencing alone. Despite this potential, these single-cell methods have seldom been applied to the study of host-associated microbial communities. Here, we present our recently developed protocols utilizing four distinct fluorescent dyes that label cells based on nucleic acid content, respiratory activity, and membrane damage. These methods have been successfully applied to study the trillions of microorganisms inhabiting the human gastrointestinal tract (the gut microbiota), in addition to a collection of isolates from five common gut-associated bacterial phyla. By merging these protocols with fluorescence-activated cell sorting and downstream multiplex 16S rRNA gene sequencing, it is possible to rapidly assess the taxonomic composition of each physiological category. These methods provide an initial step toward a robust toolkit allowing a rapid, culture-independent, and comprehensive assessment of the physiology and metabolic activity of host-associated microbial communities.

Introduction

The human body is home to trillions of microbial cells representing thousands of species (Turnbaugh et al., 2009, Qin et al., 2010) that have a profound effect on human physiology (Dutton and Turnbaugh, 2012, Haiser and Turnbaugh, 2012). These microorganisms are widely distributed among different body habitats and densely colonize the gastrointestinal tract (referred to as the gut microbiota) (Costello et al., 2009, Maurice and Turnbaugh, 2011). Recent metagenomic surveys have extensively described the structure and dynamics of the gut microbiota and its vast array of genes, the gut microbiome, in states of health and disease (Human Microbiome Project Consortium, 2012, Turnbaugh et al., 2009, Qin et al., 2010, Yatsunenko et al., 2012). However, these techniques provide limited information about metabolic activity, necessitating other methods for the quantification and identification of the active or damaged cells within host-associated microbial communities.

Flow cytometry (FCM) and cell sorting have been used in a variety of ecosystems to access the physiology of cells within complex microbial communities (Del Giorgio and Gasol, 2008, Shapiro, 2000). Combined with appropriate fluorescent dyes, FCM allows the quantification of cells with distinct levels of activity or damage, using the individual cell fluorescence and light scatter signals. In addition, the stained cells of interest can also be sorted and identified by sequencing the appropriate marker genes.

Here, we present an optimized protocol for the single-cell analysis of the human gut microbiota, validated with bacterial isolates and the intact fecal microbiota. First, we provide general information about the use of FCM and the fluorescent dyes available. We then detail our optimized protocols for characterizing the physiology of microbial cells from human fecal samples, as well as methods for the sorting and downstream 16S rRNA gene sequencing, referred to here as FACS-Seq. Finally, we discuss various experimental considerations and troubleshooting options.